Methods for diagnosis and monitoring of neurological disease by detection of an excephalotoxin

ABSTRACT

Encephalotoxin produced by activated mononuclear phagocytes is present in individuals having neurological disease including neurodegenerative and neuro-inflammatory diseases, such as Alzheimer&#39;s disease (AD), HIV-1-associated dementia (HAD), Creutzfeldt-Jakob disease, Mild Cognitive Impairment, prion disease, minor cognitive/motor dysfunction, acute stroke, acute trauma, or neuro-AIDS. Biochemical detection of encephalotoxin according to the methods of the invention will allow diagnosis of neurological disease in early, presymptomatic stages, thereby allowing early intervention in disease progression as well as identification of subjects or populations at risk for developing neurodegenerative disease. The methods of the invention also provide a mechanism for monitoring progression and treatment of neurological disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/970,587, filed Jan. 8, 2008, which is a continuation of U.S.application Ser. No. 10/543,486, now U.S. Pat. No. 7,344,853, which isthe national stage of International Application No. PCT/US2004/02236,filed Jan. 27, 2004, which claims the benefit of U.S. ProvisionalApplication No. 60/443,219, filed Jan. 27, 2003. The contents of each ofthe applications are incorporated by reference herein in their entirety.

REFERENCE TO GOVERNMENT GRANTS

This invention was made with government support under Grant No. AG12548awarded by the National Institutes of Health. The government may havecertain rights in the invention.

FIELD OF THE INVENTION

The invention relates to the correlation of clinical manifestations ofneurological disease with a neurotoxin produced by affected brainmononuclear phagocytes. The invention also relates to methods fordiagnosing a neurological disease or risk for loss of cognition bydetecting a neurotoxin in a biological sample of a subject. Theneurotoxin, encephalotoxin, has been found to be released by aninflammatory cascade that chronically damages neurons in neurologicaldisease, for example, HIV-1-associated dementia (HAD), neuro-AIDS,Creutzfeldt-Jakob Disease, Mild Cognitive Impairment, prion disease,minor cognitive/ motor dysfunction, acute stroke, acute trauma, andAlzheimer's disease (AD). The inflammatory cascade involves activationof mononuclear phagocytes and loss of synaptic connections and neurons,thus resulting in a decline in information processing, attention,learning, and information retrieval with overall loss of intellectualfunction.

BACKGROUND OF THE INVENTION

Loss of cognition and dementia associated with neurological diseaseresults from damage to neurons and synapses that serve as the anatomicalsubstrata for memory, learning, and information processing. Despite muchinterest, biochemical pathways responsible for progressive neuronal lossin these disorders have not been elucidated.

Alzheimer's disease (AD) accounts for more than 15 million casesworldwide and is the most frequent cause of dementia in the elderly(Terry, R. D. et al. (eds.), ALZHEIMER'S DISEASE, Raven Press, New York,1994). AD is thought to involve mechanisms which destroy neurons andsynaptic connections. The neuropathology of this disorder includesformation of senile plaques which contain aggregates of Aβ1-42 (Selkoe,Neuron, 1991, 6:487-498; Yankner et al., New Eng. J. Med., 1991,325:1849-1857; Price et al., Neurobiol. Aging, 1992, 13, 623-625;Younkin, Ann. Neurol., 1995, 37:287-288). Senile plaques found withinthe gray matter of AD patients are in contact with reactive microgliaand are associated with neuron damage (Terry et al., “Structural Basisof the Cognitive Alterations in Alzheimer Disease”, ALZHEIMER'S DISEASE,NY, Raven Press, 1994, Ch. 11, 179-196; Terry, R. D. et al. (eds.),ALZHEIMER'S DISEASE, Raven Press, New York, 1994; Perlmutter et al., J.Neurosci. Res., 1992, 33:549-558). Plaque components from microglialinteractions with Aβ plaques tested in vitro were found to stimulatemicroglia to release a potent neurotoxin, thus linking reactivemicrogliosis with AD neuronal pathology (Giulian et al., Neurochem.Int., 1995, 27:119-137).

Several lines of evidence now support the concept that microglia-derivedneurotoxins contribute to AD pathology. First, microglia-derived toxinscan be extracted from AD brain regions laden with plaques but not fromidentical brain regions in age-matched control or ALS brain tissues(Giulian et al. (1995) Neurochem. Int., 27: 119-137; Giulian et al.(1996) J. Neurosci., 16: 6021-6037). Second, regional distributions oftoxic activity show the greatest concentrations of microglia-derivedneuron poisons in neocortical tissues and hippocampi of AD (vs. controlsor ALS), areas containing large numbers of reactive microglia. Incontrast, cerebellum, white matter, and neocortical tissues from normalor ALS patients, which had few, if any, reactive microglial clusters,show little neurotoxic activity. Moreover, the relative number ofreactive microglial clusters in each brain region is significantlycorrelated to the level of neurotoxic activity extracted from thatregion (p<0.005). Third, isolated plaque fragments or synthetic humanAβ1-40 or Aβ1-42 peptides are found to activate human microglia torelease neurotoxins in culture (Giulian et al. (1995) Neurochem. Int.,27: 119-137; Giulian et al. (1996) J. Neurosci., 16: 6021-6037). Noneurotoxic effects, however, are detected when plaques or peptides wereplaced directly atop neurons or when microglia are exposed to fractionslacking plaques isolated from AD, ALS, or normal, aged control brains(Giulian et al. (1995) Neurochem. Int. 27: 119-137; Giulian et al.(1996) J. Neurosci. 16: 6021-6037). Thus, the toxic effects of isolatedplaques on neurons are indirect and mediated by a neurotoxic activityreleased from plaque-stimulated microglia. Fourth, there is neurotoxicactivity found in CSF from AD patients, but not detected in samples fromdisease controls (U.S. Pat. No. 6,043,283 to Giulian; Giulian et al.(1999) Am. J. Hum. Genet., 65:13-18). Fifth, infusion of Aβ-coupledmicrospheres into hippocampus produces inflammatory responses at thesite of infusion in rats (U.S. Pat. No. 6,043,283 to Giulian). Together,these data indicate that plaque-activation of microglia through contactwith Aβ peptides produces neuron-killing factors in discrete areas of ADbrain (Giulian et al. (1995) Neurochem. Int., 27: 119-137).

Although most patients developing AD will go through a transient periodof mild cognitive impairment (MCI), they will often not present to aphysician during this early phase of the disease. There is a consensusamong research groups that subjects with MCI are at increased risk forprogressing to AD (Grundman et al. (1996) Neurology 46:403; Flicker etal. (1991) Neurology 41:1006-1009; Masur et al. (1994) Neurology44:1427-1432; Tierney et al. (1996) Neurology 46:149-154). Memoryimpairment is commonly the most prominent feature of MCI but mightinclude other patterns including defects primarily in language orvisuomotor performance (Hughes et al. (1982) Br. J. Psychiatry,140:566-572; Berg (1988) Psychopharmacol. Bull., 24:637-639; Morris(1993) Neurology, 43:2412-2414; Rubin et al. (1989) Arch. Neurol.,46:379-382; Grundman et al. (1996) Neurology, 46:403; Flicker et al.(1991) Neurology, 41:1006-1009; Masur et al. (1994) Neurology,44:1427-1432;Tierney et al. (1996) Neurology, 46:149-154). Attempts atcharacterizing mild cognitive impairment have been carried out using theClinical Dementia Rating (CDR) Scale, which rates the severity ofdementia as absent, mild, moderate, or severe. Rubin et al. ((1989)Arch. Neurol., 46:379-382) concluded that individuals with a CDR of 0.5likely have “very mild” AD in the majority of cases [The CDR 0.5classification is characterized by consistent forgetfulness, which ismild with little if any impairment in other functions such asorientation, community affairs, home, and hobbies, judgment, andpersonal care.] Other measures also have been used to identify MCIsubjects. For example, poor delayed recall has been shown to be the bestpredictor of progression, the best predictor of subsequent dementia innon demented elderly subjects, and the best discriminator between normalaging and mild AD (Flicker et al. (1991) Neurology, 41:1006-1009; Masuret al. (1994) Neurology, 44:1427-1432; Tierney et al. (1996) Neurology,46:149-154). The time required for subjects with MCI to develop aclinical diagnosis of AD has been estimated by the Alzheimer's DiseaseCooperative Study (ADCS) at about 30% at 2 years and 45% at 3 years.

HIV-1 infection and neuro-AIDS produce devastating effects upon thebrain and spinal cord. Although the underlying anatomical basis forimpaired cognition during HIV-1 infection remains obscure, there is areduction of up to 40% of large neurons scattered throughout theneocortex in advanced disease with dementia (Masliah et al. (1992) J.Neuropath Exp Neurol., 51: 585-593) and a striking early loss ofsynapses (Asare et al. (1996) Am J Path 148: 31-38; Everall et al.(1993) J. Neuropath. Exp. Neurol. 52: 561-566).

HIV-1 associated dementia (HAD) is characterized by cognitivedysfunction, declining motor performance, and behavioral changes. Itoccurs primarily in the more advanced stages of HIV infection when CD4cell counts are relatively low. While the progression of dysfunction isvariable, it is regarded as a serious complication with fatal outcome.The diagnosis of cognitive loss due to HIV is by process of exclusion—noapproved marker exists to monitor HIV-specific injury to the CNS.Without such a marker, there are no clinical indications to evaluatepatients until significant functional loss appears and there are fewopportunities to develop new treatment strategies to prevent HIV braindamage. Therefore, it is very desirable to identify patients at earlypre-symptomatic stages.

Prior to HAART (defined here as combination therapy using 3 or moreanti-retroviral agents), 60% of those with AIDS developed dementia. Thisincidence appears to have fallen to about 10 to 15%, but cognitivedysfunction remains a problem for over half of the HIV/AIDS population(Giulian et al. (1990) Science, 250: 1593-1596; Giulian et al. (1993)Proc. Natl. Acad. Sci., 90:2769-2773; Giulian (1995) In: NEUROGLIA (IIKettenmann, B Ransom Eds) Oxford University Press, pp. 671-684; Giulianet al. (1998) In: INFLAMMATORY MECHANISMS OF NEURODEGENERATION AND ITSMANAGEMENT (P. Wood, ed.); Humana Press, Vol 4, pp. 109-128).

HIV-1 brain pathology involves diffuse synaptic damage in the neocortex,the loss of cortical neurons, and a population of infected, reactivemononuclear phagocytes, including invading blood monocytes, microglia,and multi-nucleated giant cells. These giant cells represent a fusion ofHIV-infected mononuclear phagocytes that are coated with gp120, theretroviral envelope protein; presence of giant cells has been correlatedwith cognitive impairment during HIV-1 infection. Currently, mostresearch groups in the field agree that poisons released by infectedmononuclear phagocytes are a primary cause of cognitive loss in theHIV-1(+) population (Vitokovic et al. (1998) Medical Sciences, 321:1015-1021; Morgello et al. (2001) Neuropath. App. Neurobiol., 27:326-335; Lawrence et al. (2002) Microbes and Infection, 4: 301-308;Masliah et al. (1992) J. Neuropath. Exp. Neurol., 51: 585-593; Maslliahet al. (1995) J. Neuropath. and Exp. Neurol., 54: 350-357; Asare et al.(1996) Am. J. Path., 148: 31-38; Everall et al. (1993) J. Neuropath.Exp. Neurol., 52: 561-566).

Several lines of evidence now support the concept that mononuclearphagocyte-derived neurotoxins contribute to the neuron injury withinbrain during HIV-1 infection. First, HIV-1 neither infected neurons norshowed a direct toxic effect upon neurons (Giulian et al. (1996) J.Neurosci., 16:3139-3153, Giulian et al. (1990) Science 250: 1593-1596;Levine et al. (1976) Biochim. Biophys. Acta, 452: 458-467). Second,HIV-1 mononuclear phagocytes (THP-1, U937, human blood monocytes, andhuman brain microglia) released neurotoxins when infected in vitro withHIV-1; in contrast, lymphocytes (H9, human blood lymphocytes) did not(Giulian et al. (1996) J. Neurosci., 16:3139-3153; Giulian et al. (1990)Science, 250: 1593-1596). Third, human mononuclear phagocytes (bloodmonocytes and microglia) isolated from infected donors released the sameneurotoxin as recovered from in vitro experiments; again, isolatedinfected lymphocytes did not (Giulian et al. (1996) J. Neurosci.,16:3139-3153). Fourth, neurotoxic activity can be recovered from braintissues of infected individuals (Giulian et al. (1993) Proc. Natl. Acad.Sci., 90:2769-2773; Giulian (1995) In: NEUROGLIA (H Kettenmann, BRansom, Eds,) Oxford University Press, pp. 671-684; Giulian et al.(1998) In: INFLAMMATORY MECHANISMS OF NEURODEGENERATION AND ITSMANAGEMENT (P. Wood, ed.); Humana Press, Vol 4, pp. 109-128). Fifth,gp120, the viral envelope glycoprotein, can stimulate neurotoxin releasefrom human blood monocytes and microglia; other viral proteins includingthat did not (Levine et al. (1976) Biochim. Biophys. Acta, 452:458-467). Sixth, high concentrations of neurotoxin were found in thecerebrospinal fluid of HIV-1(+) individuals. And seventh, a family ofneurotoxic heparan oligosaccharides can be isolated from HIV-1 infectedcells and from HIV CSF.

Although reactive mononuclear phagocytes release a number of bio-activesubstances, few of these compounds are actually able to harm neurons atconcentrations found to exist in neurodegenerative disease (Hardy et al.(2002) Science, 297:353; Mourdian et al., (1989) Neurosci. Lett., 105:233; Milstein et al. (1994) J. Neurochemistry, 63, 1178; Giulian et al.(1990) Science, 250:1593). Moreover, few of such candidate neuronpoisons are present in both AD and HAD. For example, increased tissueconcentrations of “toxic” forms of Aβ1-42 are characteristic for AD(Hardy et al. (2002) Science, 297:353), but do not occur in HAD.Similarly, elevated quinolinic acid levels occur in the cerebrospinalfluid (CSF) of subjects with HAD (Mourdian et al. (1989) Neurosci.Lett., 105:233), but not in those with AD (Milstein, et al. (1994) J.Neurochemistry, 63: 1178). In contrast, both AD and HAD brain tissuescontain a heterogeneous group of small stable molecules with potentneurotoxic actions (Giulian et al. (1990) Science, 250:1593; Giulian etal. (1995) Neurochem. Int., 27:119; Giulian et al. (1996) J.Neuroscience 16: 6021). Cultured mononuclear phagocytes activated byexposure to amyloid plaques, synthetic β-amyloid peptides, HIV-1, orgp120, produce these same neurotoxins (Giulian, et al. (1993) Proc.Natl. Acad. Sci. USA, 90: 2769; Giulian et al. (1998) J. Biol. Chem.,273: 29719). Such observations suggest that a common, thoughunidentified, pathway mediates immune-driven neuron pathology in both ADand HAD.

As the clinical expression of neurological disease may occur only aftera significant degree of neuron loss and synaptic damage beyond acritical threshold necessary for adequate adaptive function, earlypre-symptomatic detection of disease pathology offers the opportunity toslow disease progression. The present invention provides methods fordiagnosis of neurological disease and risk for loss of cognition,including, for example, Alzheimer's disease, HIV-1 associated dementia(HAD), neuro-AIDS, Creutzfeldt-Jakob disease, Mild Cognitive Impairment(MCI), prion disease, minor cognitive/ motor dysfunction, acute stroke,or acute trauma. The methods of the invention allow early detection ofneurological disease and risk for loss of cognition, thereby allowingearlier intervention in the progression of disease. Also provided aremethods for monitoring the progression and treatment of neurologicaldisease by monitoring encephalotoxin levels in a subject.

SUMMARY OF THE INVENTION

The present invention provides various embodiments of methods fordiagnosis of neurological disease or risk for loss of cognition in asubject. This is accomplished by detecting an encephalotoxin in abiological sample of the subject. In some embodiments of the invention,detection of the encephalotoxin involves contacting a biological sampleof the subject with neurons both in the presence of and in the absenceof an inactivator of the encephalotoxin and comparing neuron survival inthe presence of the encephalotoxin inactivator relative to neuronsurvival in the absence of the encephalotoxin inactivator. A decrease inneuron survival in the absence of the encephalotoxin inactivator isindicative of the neurological disease or risk for loss of cognition. Insome embodiments of the invention, encephalotoxin is detected bymeasuring light absorbance of the biological sample in the both thepresence of and in the absence of a encephalotoxin inactivator, anincrease in absorbance in the absence of the encephalotoxin inactivatorbeing indicative of neurological disease or risk for loss of cognition.Preferably, light absorbance is measured at a wavelength of 232nanometers (nm).

In some embodiments of the invention, the encephalotoxin is anoligosaccharide having at least one glucosamine having N-sulfation andO6-sulfation; lacking peptide bonds; and having a molecular mass of lessthan about 2000 daltons. Preferably, the encephalotoxin has 4 to 8saccharide units. Preferably, the molecular mass of the encephalotoxinis between about 700 and 1900 daltons.

In some embodiments, the encephalotoxin inactivator is heparin lyase I,nitrous acid, glucosamine-6-sulfatase, or N-sulfamidase. Preferably, thenitrous acid solution has a pH of about 1.5.

In some embodiments of the invention, the biological sample iscerebrospinal fluid, spinal cord tissue, or brain tissue.

Neurological diseases that may be diagnosed or monitored by the methodsof the invention include neurodegenerative and neuro-inflammatorydiseases and disorders such as, but not limited to, Alzheimer's Disease,Creutzfeldt-Jakob Disease, Human Immunodeficiency Virus-1(HIV-1)-associated dementia (HAD), Mild Cognitive Impairment (MCI),prion disease, minor cognitive/ motor dysfunction, acute stroke, acutetrauma, and neuro-AIDS. In various embodiments, the methods of theinvention may be used in the diagnosis or monitoring of human, primate,bovine, equine, canine, feline, porcine, or rodent subjects.

In some embodiments of the invention, comparison of neuron survivalcomprises comparison of the ED₅₀ of the encephalotoxin in the presenceof the encephalotoxin inactivator relative to the ED₅₀ of theencephalotoxin in the absence of the encephalotoxin inactivator, whereina lower ED₅₀ of the encephalotoxin in the absence of encephalotoxininactivator relative to the ED₅₀ of the encephalotoxin in the presenceof encephalotoxin inactivator is indicative of neurological disease orrisk for loss of cognition.

In further embodiments of the invention are provided methods ofmonitoring treatment of a neurological disease in a subject. In someembodiments, the method of monitoring involve comparing theencephalotoxin levels in a first and second biological sample of asubject, wherein the first biological sample is taken from the subjectat an earlier timepoint than the second biological sample, wherein thesecond biological sample is taken from the subject following treatmentof the neurological disorder, and wherein encephalotoxin level ismeasured by light absorbance of the biological sample, an increase inabsorbance of the second biological sample being indicative ofprogression of the neurological disease. In some embodiments, the firstbiological sample is taken, removed, or extracted from the subjectfollowing a treatment (e.g., administration of a drug) of theneurological disease.

In further embodiments of the invention are provided methods ofmonitoring progression of neurological disease in a subject comprisingdetecting an increase in encephalotoxin level in said subject over time,wherein detecting the increase in encephalotoxin level comprisesmeasuring an increased light absorbance of an encephalotoxin in a firstbiological sample of the subject relative to light absorbance of anencephalotoxin of a second biological sample of the subject, wherein thesecond biological sample is taken from the subject before the firstbiological sample, increased light absorbance being indicative ofprogression of the neurological disease.

Also provided by embodiments of the invention are methods for monitoringprogression of neurological disease in a subject comprising detecting anincrease in encephalotoxin level in the subject over time, whereindetecting the increase involves contacting a first biological sample ofthe subject with neurons, contacting a second biological sample of thesubject with neurons, and detecting decreased neuron survival in thepresence of the second biological sample, wherein the second biologicalsample is taken at a later timepoint than the first biological sample;and wherein decreased neuron survival in the presence of the secondbiological sample is indicative of progression of the neurologicaldisease.

In some embodiments of the invention, one of the biological samples istaken during the prodromic phase of said neurological disease.

In another embodiment of the invention, methods of monitoring treatmentof a neurological disease in a subject by detecting an increase inencephalotoxin level in a subject over time, wherein detecting theincrease in encephalotoxin level involves contacting a first biologicalsample of the subject with neurons, contacting a second biologicalsample of the subject with neurons, and detecting decreased neuronsurvival in the presence of the second biological sample, wherein thesecond biological sample is taken at a later timepoint than the firstbiological sample and following a treatment of the neurological disease;and wherein decreased neuron survival is indicative of progression ofthe neurological disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates inactivation of encephalotoxin by various methodsspecific for heparan sulfate and heparin. As shown in FIG. 1A,encephalotoxin released by BV2 microglia was inactivated by nitrous acidpH 1.5, by heparin lyase I (E.C. 4.2.2.7), and by sulfatases that cleaveat O-6 and from N-sulfated glucosamine (GlcNS) (glucosamine-6-sulfatase(E.C. 3.1.6.14) and N-sulfaminidase (E.C. 3.10.1.1)). As shown in FIG.1B, encephalotoxin found in ventricular CSF of AD brain was inactivatedby nitrous acid pH 1.5, by heparin lyase I, and by sulfatases thatcleave at O-6 and from GlcNS. As demonstrated in FIG. 1C, encephalotoxinrecovered from lumbar CSF of subject with AD was inactivated by nitrousacid pH 1.5, by heparin lyase I, and by sulfatases that cleave at O-6and from GlcNS.

FIG. 2 illustrates the determination of molecular mass of encephalotoxinusing a TSK-GW2500PXL with a linear sieving range from 300 to 3000daltons. Commercially available heparan oligomers were used asstandards. CSF samples (100 μl) from probable AD showed a minor peak andmajor peak of neurotoxic activity that range in size from about 700 to1,900 daltons. These estimated molecular masses suggest that at leastsome forms of encephalotoxin comprise about 4 to 8 saccharide residues.

FIG. 3 shows dose response curves for encephalotoxin isolated fromprobable AD, MCI, and normal elderly subjects. Increasing amounts oftoxin are found in those subjects with greater cognitive impairment.

FIG. 4A shows the results of anion-exchange HPLC (ProPAK PA1, 0.0 to 0.7M NaCl, UV @ 232 nm) separation of encephalotoxin from microglial BV2cells stimulated with Aβ1-42. Three peaks (PEAK 38, 48, and 53)corresponding to the encephalotoxin were detected. The encephalotoxin ofPEAKS 38, 48, and 53 was 1) sensitive to heparin lyase I, 2) sensitiveto nitrous acid pH 1.5 and 3) toxic to hippocampal neurons (data notshown). As demonstrated in FIG. 4B, these same peaks were absent fromconditioned media recovered from control BV2 cells that were not exposedto Aβ1-42.

FIG. 5A illustrates the presence of encephalotoxin in ventricular andlumbar CSF recovered from autopsy cases. FIG. 5B illustrates thepresence of encephalotoxin in ventricular and lumbar CSF recovered fromliving subjects. Data are expressed in terms of CSF volumes required toelicit death of cultured hippocampal neurons. As shown in the doseresponse curves (FIG. 5A), small volumes of high toxin concentrationsshift curves to the left, as found in those subjects with definite AD(diagnosis confirmed by autopsy). These data can also be expressed asED₅₀s (volumes of CSF required to give 50% maximal killing) As shown inFIG. 5B, a similar pattern was found in those subjects with probable AD(clinical diagnosis) who have small ED₅₀s (0.1 to 10 μl), followed bythose in the MCI group with moderate values (10 to 200 μl). Importantly,various other diagnostic groups showed no detectable encephalotoxin(ED₅₀s>1000 μl).

FIG. 6 shows PEAKs 38, 48, and 53 in CSF of AD (Panels A,B) and MCI (C),but not in normal elderly control (D) in anion exchange HPLC. Thesepeaks were heparin lyase I sensitive (data not shown). As shown in FIG.6E, bioassays of these HPLC fractions confirm the same peaks areneurotoxic.

FIG. 7 shows that, in anion-exchange HPLC (linear gradient of 0 to 2.0 MNaCl over 90 min), 3 discrete peaks of neurotoxic activity are found in100 μl of CSF from definite AD (FIG. 7A), probable AD (FIG. 7B), and HAD(FIG. 7C). No toxic activity is recovered from vascular dementia (FIG.7A). Heparin lyase I and N-sulfaminidase, but not heparin lyase II,eliminate all toxin peaks.

FIG. 8 illustrates the CSF Neurotoxicity Index, [calculated as value ofequivalent volume of CSF to yield 50% of total killing effect upon astandardized rat hippocampal neuron culture assay] from cerebrospinalfluid (CSF) samples from a variety of neurological disorders. As shownin FIG. 8A, samples from definite Alzheimer's disease (AD) and HIV-1infection contain encephalotoxins. Cerebrospinal fluid obtained duringroutine lumbar myelogram (Myelograms) were from subjects without memorycomplaints. Neuropathy refers to subjects with cranial or peripheralnerve disorders while subjects with psychiatric diagnoses had noevidence of neurological disease. Other neurological diseases includedfungal meningitis, neuro-syphilis, multiple sclerosis (MS), andamyotrophic lateral sclerosis (ALS). FIG. 8B compares CSF index scoreswith HIV-1(+) volunteers with no cognitive loss, mild cognitive motordysfunction (MCMD), or HAD. Significant differences exist among MCMD andHAD, again supporting the pattern that more toxin is associated withgreater degrees of cognitive impairment. FIG. 8C compares CSF indexscores for elderly volunteers with no cognitive loss, with MCI, withprobable AD, or with non-AD dementia (caused by traumatic, vascular, orethanol injury). MCI shows a consistent and significantly elevated levelof encephalotoxin above other forms of dementia. Bars show medianvalues. FIG. 8D compares Neurotoxicity Index values vs. T scores for thepaced auditory serial-addition test (PASAT, a sensitive measure ofinformation processing.) As shown, a significant linear relationshipexists between CSF Neurotoxicity Index and this cognitive measure (n=26;p<0.0001; correlation coefficient=0.74).

FIG. 9 shows a comparison of CSF Neurotoxicity Index scores of CSF fromelderly subjects. Probable AD and MCI show significant toxin levels withan overlap in distribution of values. CSF neurotoxin levels clearlyseparate AD pathology from other categories common to the aged.(Bar=mean values.)

FIG. 10 shows two examples of drug effects upon CSF encephalotoxinlevels. Single drug treatment (identity of drugs remains coded) failedto offer full suppression of toxin (i.e., shifted Index scores to anormal range of >100 as noted in Table 1) after a 6-week trial. Incontrast, DAP/HCQ for 6 weeks provided complete inhibition of toxinproduction in all subjects tested to date (5 of 5).

The referenced patents, patent applications, and scientific literaturereferred to herein are hereby incorporated by reference in theirentirety. Any conflict between any reference cited herein and thespecific teachings of this specification shall be resolved in favor ofthe latter. Likewise, any conflict between an art-understood definitionof a word or phrase and a definition of the word or phrase asspecifically taught in this specification shall be resolved in favor ofthe latter.

As used herein, the term “about” refers to an approximation of a statedvalue within an acceptable range. Preferably the range is +/−10% of thestated value.

Definite AD was diagnosed at autopsy using consensus neuropathologicalcriteria (The NIA-Reagan Working Group. Consensus recommendations forthe postmortem diagnosis of Alzheimer's disease. (1997) Neurobiol.Aging, 18:S1). The clinical definition for probable AD followedconsensus recommendations (McKhann et al. (1984) Neurology 34:939) withimpairment defined as psychometric performance falling at least 2standard deviations (SD) below mean normative mean values inLearning/Memory [measured by the Wechsler Memory Scale-III LogicalMemory Subtest, Hopkins Verbal Learning Test-Revised, or Brief VisualMemory Test-Revised, and 2 SD below normative mean on at least one testwithin the following cognitive domains: Attention/Information Processing[Verbal Sustained Attention Test, Symbol Digit Modalities Test, WechslerAdult Intelligence Test-III Digit Span, Trails A Test, and PacedAuditory Serial-Addition Test (PASAT)], Orientation (Orientationquestions), Language [Naming and Category Fluency, FAS Test], ExecutiveFunction [Wisconsin Card Sort Test and Trials B Test]. Subjects with MCIare defined as those without dementia but who show amnestic featuresincluding a memory complaint confirmed by an informant and a memoryimpairment measured at least 1.5 SD below normative mean values usingthe same testing battery as for AD.

The clinical definitions for HIV-related cognitive impairments followedconsensus recommendation (Working Group of American Academy of NeurologyAIDS Task Force (1992) Neurology, 41:778) with subjects showing noevidence for other etiologies. Measured impairment for HIV-associateddementia (HAD) fell 2.5 SD below normative means in one domain or 2 SDin at least two domains on any of the following tests: Learning/Memory,Language, Attention/Information Processing, Abstraction/Problem Solving,and Motor Abilities [Grooved Pegboard]. Subjects with mildcognitive-motor dysfunction (MCMD) are defined as those falling 1.5 SDbelow mean normative values in any test in at least two cognitivedomains or 2.0 SD below mean values in a single domain.

As used herein, “loss of cognition” or variants thereof refer to adecline in at least one of information processing, attention, learning,information retrieval, and overall loss of intellectual function. Lossof cognition may be measured by any method known in the art, including,for example, Attention/Information Processing [Verbal SustainedAttention Test, Symbol Digit Modalities Test, Wechsler AdultIntelligence Test-III Digit Span, Trails A Test, and Paced AuditorySerial-Addition Test (PASAT)], Orientation (Orientation questions),Language [Naming and Category Fluency, FAS Test], Executive Function[Wisconsin Card Sort Test and Trials B Test], Learning/Memory,Abstraction/Problem Solving, Motor Abilities [Grooved Pegboard], andHopkins Verbal tests. A subject at risk for loss of cognition has nomeasurable loss of cognition but has a greater chance for loss ofcognition than the average population. For example, a first-degreerelative of an Alzheimer's disease patient is at risk for loss ofcognition.

As used herein, the term “contact” or “contacting” means bringingtogether, either directly or indirectly, a compound into physicalproximity to a molecule of interest. Contacting may occur, for example,in any number of buffers, salts, solutions, or in a cell or cellextract.

The term “peptide bond” means a covalent amide linkage formed by loss ofa molecule of water between the carboxyl group of one amino acid and theamino group of a second amino acid.

The term “saccharide” or “saccharide unit” includes oxidized, reduced orsubstituted saccharides. Saccharides of this invention include, but arenot limited to, ribose, arabinose, xylose, lyxose, allose, altrose,glucose, mannose, fructose, gulose, idose, galactose, talose, ribulose,sorbose, tagatose, gluconic acid, glucuronic acid, glucaric acididuronicacid rhamnose, fucose, N-acetyl glucosamine, N-acetyl galactosamine,N-acetyl neuraminic acid, sialic acid, N-sulfated glucosamine (GlcNS),2-sulfated iduronic acid (IdoA2S), derivatives of saccharides such asacetals, amines, and phosphorylated sugars, oligosaccharides, as well asopen chain forms of various sugars, and the like. “Oligosaccharide”refers to a molecule having two or more saccharide units.

The term “purified”, when used to describe the state of the neurotoxinof the invention, refers to a neurotoxin substantially free of othercellular material. “Substantially free” refers to at least about 60% orabout 70%, more preferably at least about 80% or about 90%, and mostpreferably at least about 95%, about 98%, or about 100% free of othercellular materials.

The prodromic phase of pathology of neurodegenerative orneuro-inflammatory disease is defined herein as that stage in thedisease during which the clinical manifestations of cognitive,behavioral, or social impairment have not yet reached a diagnosticthreshold for MCI (amnesic features with memory testing 1.5 SD belownormative mean) or AD (2 SD below normative means in memory and at leastone other cognitive domain). By this definition, the prodromic phasewould encompass, for example, the clinically-defined CognitiveImpairment, No Dementia (CIND) population (Toukko et al. (2001) Int.Psychogeriatr., Supp. 1:183-202), the at-risk asymptomatic populationdescribed by Hom ((1994) J. Clin. Exp. Neurol., 16: 568-576), theAge-Associated Memory Impairment (AAMI, Goldman et al. (2001) Alz. Dis.Assoc. Dis., 15: 72-79), the subclinical cohort of the Farmington study(Elias et al. (2000) Arch. Neurol. 57: 808-813) or a preclinical ADpopulation defined identified at autopsy (Price et al. (2001) Arch.Neurol., 58: 1395-1402). In general, all these groups show memory testvalues (verbal, episodic memory) at about 1 SD below normative meanscores adjusted for age, education, and ethnicity. Overall, populationsat-risk for AD showed longitudinal declines at a rate of about 0.3 to0.6 SD per year from normalized memory test scores tests.

The signaling cascade involved in the neurodegenerative diseasesaddressed by the present invention comprises events including (1)mononuclear phagocyte activation; (2) mononuclear phagocyte release ofencephalotoxin; and (3) the toxic effect of encephalotoxins on neurons.Neurotoxicity of a mononuclear phagocyte induced by a mononuclearphagocyte activator may be inhibited or inactivated by an agent referredto herein as a neurotoxin inhibitor or inactivator.

A mononuclear phagocyte is an immune cell which has a single nucleus andthe ability to engulf particles, also known as phagocytosis. Mononuclearphagocytes are found in blood and body tissues, including the centralnervous system and brain, and include, for example, microglia cells,monocytes, macrophages, histiocytes, dendritic cells, precursor cells ofmicroglia, precursor cells of monocytes, precursor cells of macrophages,microglia-like cell lines, macrophage-like cell lines, or cell linesmodified to express microglia-like surface molecules that are active inaccordance with the above definition of mononuclear phagocyte. A neuronas defined in accordance with the present invention includes a neuronand neuron-like cell, which is a cell modified to express aN-methyl-D-aspartate receptor which neuron exhibits neuronal activityunder typical normal, non-diseased state, conditions.

Mononuclear phagocyte activation initiates a process that causes therelease of neurotoxins. Mononuclear phagocyte activation is alsoreferred to herein as immune activation, markers of which are anyprocess that renders a mononuclear phagocyte more dynamic andcharacterized by activities such as and not limited to increasedmovement, phagocytosis, alterations in morphology, and the biosynthesis,expression, production, or secretion of molecules, such as protein,associated with membranes including complement, scavengers, Aβ, andblood cell antigens, histocompatibility antigens for example. Productionof molecules includes enzymes involved in the biosynthesis of bioactiveagents such as nitric oxide synthetase, superoxide dismutase, smallmolecules such as eicosanoids, cytokines, free radicals and nitricoxide. Release of factors includes proteases, apolipoproteins such asapolipoprotein E, and cytokines such as interleukin-1, tumor necrosisfactor as well as other molecules such as encephalotoxin and hydrogenperoxide.

Mononuclear phagocyte neurotoxicity or neuron toxicity refers to aprocess that leads to the injury, destruction, or death of neurons,which is measured by loss of metabolic function, release ofintracellular material, penetration of impermeant dyes, reduction ofcell number measured by biochemical or histological methods. Forexample, changes in biochemical markers such as loss of neurofilamentsor synaptophysin or release of lactate dehydrogenase, or other evidenceof cell injury such as penetration of impermanent dyes, includingfluorescent nuclear dyes and trypan blue. These and other strategies foridentifying cell injury, destruction or death, or measuring neuronfunction, are known to one skilled in the art and are contemplated bythe present invention.

Neurotoxin is defined herein as a substance that injures, damages, orkills a neuron while sparing other central nervous system cells such asglia, for example. A neurotoxin interacts with neurons in such a way asto disrupt neuron function and survival. The possible actions of aneurotoxin on neurons, also referred to herein as neuronal damage,include inhibition or disruption of normal cell metabolism, includingmetabolism of glucose, the production of ATP, and maintenance of iongradients across cell membranes including Na⁺, Ca²⁺, and K⁺ ionchannels, the synthesis of proteins and nucleic acids, and mitochondrialrespiration, and cell death.

Encephalotoxin as used herein refers to a class of neurotoxins havinglow molecular mass (<2000 daltons), heat stability, resistance toproteases, and loss of activity upon exposure to nitrous acid,N-sulfamidase, glucosamine-6-sulfatase, and heparin lyase I.Encephalotoxins comprise at least one GlcNS residue. An encephalotoxinpreferably has a molecular weight between about 700 and 1,900 daltons.The encephalotoxin preferably has 4 to 8 saccharide residues.

Encephalotoxin inactivators or inhibitors are agents which inactivateneurotoxin or inhibit the effects of neurotoxins that are released fromactivated mononuclear phagocytes. For purposes of the present invention,inhibit, inhibition, inactivate, inactivation, and variations thereofare used synonymously with reduce, suppress, retard, slow, and suspend.Inactivation or inhibition also refers to complete inhibition of theneurotoxin cascade such that the cascade is arrested, stopped, orblocked. Encephalotoxin inactivation includes reduction of neurotoxicactivity by about 10%, 20%, 50%, more preferably about 80%, 90%, or 95%,and most preferably about 98%, 99%, or 100%. By way of example, acompound is an encephalotoxin inactivator if it reduces the neurotoxicactivity of the encephalotoxin or increases neuron survival such thatneurons otherwise at risk of damage upon exposure to the encephalotoxinare not damaged in the presence of the encephalotoxin and the compound.Preferably, more than about 10%, 20%, or 50% of the neurons at risk arenot damaged by the encephalotoxin in the presence of the encephalotoxininactivator. Even more preferably, about 80%, 90%, or 95%, and mostpreferably, about 98%, 99%, or 100% of the neurons at risk are notdamaged by the encephalotoxin in the presence of the encephalotoxininactivator. Preferable encephalotoxin inactivators of the inventioninclude heparin lyase I, N-sulfaminidase, glucosamine-6-sulfatase, andnitrous acid. Nitrous acid preferably has a pH of about 1.5. Morepreferably, exposure to nitrous acid occurs at room temperature.

An effective amount of a mononuclear phagocyte and an activator is theamount of each normally resulting in an event in the cascade, but forthe addition of an encephalotoxin inactivator. An effective amount willbe known to a skilled artisan in view of the present disclosure and willvary depending on the use of a mononuclear phagocyte, neuron, activatoror components, and the mammalian origin of the cells.

In vitro neurotoxicity assays of the invention detect the presence ofencephalotoxin and inactivation thereof and employ cultures of neuronsor neuron-like cell lines which have been modified to expressN-methyl-D-aspartate receptors. The presence of neurotoxic activity, ora measure of neuron function or measure of neuron survival, will bedetermined by reduction in cell number, changes in biochemical markerssuch as loss of cell metabolic function, release of intracellularmaterial, penetration of impermeant dyes, such as and not limited tofluorescent nuclear dyes and trypan blue, loss of neurofilament orsynaptophysin, release of lactate dehydrogenase, or other evidence ofcell injury. Other methods of measuring neuron function includedetecting the inhibition of normal cell metabolism including thedisruption of glucose metabolism, ATP production, ion gradientmaintenance across cell membranes, and protein synthesis, nucleic acidsynthesis, and mitochondrial respiration. Reductions in an inflammatorymarker or injury to a neuron by a test biological sample may be comparedto a control. These and other strategies for identifying cellneurotoxicity or measuring neuron function, which may be displayed ascell injury, are known to one skilled in the art and are contemplated bythe present invention.

Using the assay systems of the invention, it is possible to diagnosesubjects at early, for example, pre-symptomatic or prodromic, stages ofneurological disease. It is further possible, using the methods of theinvention, to identify subjects or populations at risk for loss ofcognition by detecting the encephalotoxin in a biological sample of asubject. The methods of the invention also allow monitoring ofprogression of neurological disease by detecting increases inencephalotoxin levels of a subject over time. The patients or subjectsto be diagnosed in accordance with the present invention include and arenot limited to mammals such as humans, primates such as and not limitedto monkey, chimpanzee, and ape, rodents, such as and not limited to ratand mouse, guinea pig, dog, cat, rabbit, and pig. Biological samples inaccordance with the methods of the invention include central nervoussystem tissue, such as brain or spinal cord tissue, or cerebrospinalfluid (CSF). The neurological diseases to be identified or monitoredaccording to the invention include neurodegenerative andneuro-inflammatory diseases such as, but not limited to, Alzheimer'sdisease, Creutzfeldt-Jakob disease, HIV-1 associated dementia (HAD),Mild Cognitive Impairment, prion disease, minor cognitive/ motordysfunction, acute stroke, acute trauma, neuro-AIDS, and immune-mediatedbrain inflammation.

The methods of the present invention include a neurotoxin assay of abiological sample of a patient, which can be used to diagnose aneurological disease or disorder or risk for loss of cognition in thesubject. The methods of the present invention also may be used as anearly detection method to identify individuals who are at risk fordeveloping neurological diseases or disorders in view of their age,family history, early symptoms or other risk factors. For example, abiological sample, such as blood, spinal cord tissue, cerebrospinalfluid, or brain tissue, may be taken from a patient and evaluated withthe encephalotoxin inactivators of the present invention, as describedherein, to identify the presence of encephalotoxins in the patient or toidentify patients who may suffer from a neurological disease. Thepatient's sample may be compared to a control to determine whetherelevated levels of neurotoxins are present.

Similarly, the methods of the present invention employ the neurotoxininactivators of the invention to monitor a patient's treatment or therate of progression of a disease by determining the amount ofneurotoxins that are present in the patient's system before andthroughout treatment. The methods may also be used to monitor neurotoxinlevels to allow for the adjustment of drug doses.

For example, the present invention provides methods for assaying thepresence and level of encephalotoxin in a patient by contacting abiological sample of the patient with an encephalotoxin inactivator,such as heparin lyase I, N-sulfaminidase, glucosamine-6-sulfatase, ornitrous acid. Thereafter, the amount of inhibition in the presence ofthe inactivator is compared to a measured control. There is an increaseof encephalotoxin in the subject when there is an increase in theencephalotoxin level compared to the control.

The present invention offers strategies for early detection ofneurodegenerative disease or risk for loss of cognition, therebyallowing early intervention in disease progression. The followingexamples are illustrative only and are not intended to limit the scopeof the invention.

EXAMPLES

Purification of Encephalotoxin

Encephalotoxins were isolated from cerebrospinal fluid by HPLC sievingchromatrophy (TSK-GEL G2500PWXL column; 7.8×300 mm from TosohBioscience; Montgomeryville, Pa.) eluted with 2 M NaCl; by anionexchange HPLC (tandem ProPac PA1 columns 4×250 mm from Dionex Corp.;Sunnyvale, Calif.) with a linear gradient of 2 M NaCl over 180 min; orby adsorption chromatography (Oasis Cartridges, Waters) using themanufacturer's protocol.

Structural Characterization and Inactivation of Encephalotoxin

Structural characterization and inactivation of encephalotoxin (isolatedby organic extractions, gel filtration, and sequential C18 HPLC fromAβ-stimulated microglial cell line BV2) was performed by various nitrousacid cleavage protocols. Neurotoxic activity was eliminated by nitrousacid treatment at pH 1.5 but not by other acid treatments at pH 4.0 orwith hydrazinolysis (FIG. 1). The results indicated that the internalstructure of encephalotoxin contained at least one GlcNS residue.Encephalotoxin chemical structure was further examined by treatmentswith highly selective enzymes that attack heparin or heparan sulfate(HS) polymers. Traditionally, heparin lyase I acts primarily onheparin-containing GlcNS(1→4)IdoA2S sequences and heparin lyase III onHS primarily at a GlcNAc(1→4)IdoA or GlcNAc(1→4)GlcA sequence.(Generally, these enzymes require oligomers of at least 4 residues.)Finally, encephalotoxin was treated with sulfatases that are highlyselective for O-sulfation sites at positions 2, 3, or 6 (found in HS andheparins) as well as N-sulfamidase which cleaves the N-sulfation site(FIG. 1). Heparin lyase I [GlNS(1→4)IdoA2S], but not heparin lyase III,inactivated encephalotoxin as did sulfatases that removed groups fromO-6S and GlcNS. Additionally, chemical methods to modify terminal amines(acetylation, PFPA modification, etc.) suggested the presence ofterminal amines, such as unsubstituted GlcN residues. Accordingly,encephalotoxin contains heparin-like oligosaccharides of at least 4residues with GlcNS, IdoA2S, GlcN residues plus O-linked sulfation atposition 6.

Molecular mass of the neurotoxin was estimated using a TSK-GW2500PXLwith a linear sieving range from 300 to 3000 daltons. Commerciallyavailable heparan oligomers were used as standards. CSF samples (100 ul)from probable AD showed a minor peak and major peak of neurotoxicactivity having low molecular weight ranging in size from about 700 to1,900 daltons. These estimated molecular masses suggest oligosaccharidesfrom about 4 to 8 residues in length (FIG. 2).

Neurotoxin Bioassay

Cultured neurons prepared from rat hippocampus were used in toxicitystudies. These cultures consist of process-bearing neurons (10-20% oftotal cell population) atop a bed of astroglia (>70%) mixed withmicroglia (5-10%). In order to eliminate microglia, cultures wereexposed to saporin coupled to acetylated LDL at 10 μg/ml for 18 hours.At the end of 72 hrs, the cultures were fixed in 3% paraformaldehyde atroom temperature for 12 hours and immuno-stained by overnight incubationwith a mixture of anti-neurofilament antibodies (SMI-311, 1:150; RT-97,1:150; Sternberger Monoclonals, Inc.;) plus anti-MAP-2 (1:200;Boehringer Mannheim, 184959;) at 4° C. in the presence of 2% horse serumand 0.3% Triton X-100 to delineate both neuronal cell bodies andneurites. Immuno-labeled cells per field were scored at 200×magnification using fluorescence microscopy. Neuron killing wasexpressed as % mean survival expressed in terms of parallel untreatedcontrol cultures after scoring at least 8 randomly selected fields foreach of 3 coverslips.

1 ml of CSF was fractionated by adsorption chromatography, dried undervacuum, and reconstituted in artificial CSF comprising electrolytes,such as NaCl, and glucose. Increasing amounts of fractionated toxin(range 0.1 to 500 ul equivalents of original sample volume) were addedto triplicate cultures. Results were plotted as volume vs. % neuron kill(with kill calculated as % loss of immuno-stained hippocampal neuronsagainst untreated control cultures). Inactivation, for example, byheparin lyase I, N-sulfaminidase, glucosamine-6-sulfatase, or nitrousacid treatment, was used to confirm the presence of encephalotoxin foreach CSF sample tested. As shown in FIG. 3, high levels of toxin (curvesshifted to left) for AD, intermediate levels (curve shifted to right)for MCI, and toxin-free (flat line) profiles were noted for samplestaken from disease controls. In order to compare different populations,a CSF Neurotoxicity Index was developed to assign scores that reflectlevel of neurotoxin. This index was calculated as an ED₅₀ (theequivalent CSF volume that yields 50% of the maximal level of neuronkilling) Using this measurement, high neurotoxin levels have low Indexscores; for example, high toxin concentrations have low Index scores ofabout 1, intermediate levels at about 5 to 100, and normal elderly showvalues of 1000.

Encephalotoxin Chemical Assay

Anion-exchange HPLC conditions for the detection of encephalotoxin wereestablished (0.0-0.7 m NaCl gradient; ProPAK PA-1 column; 232 nm UVmonitoring). The microglial cell line BV2 was exposed to human Aβ1-42for 48 hr and the conditioned media fractioned by adsorptionchromatography. Three biologically-active peaks (PEAKs 38, 48, and 53)were recovered that corresponded to 3 peaks detected by 232 nm (FIG.4A). All 3 peaks were sensitive to nitrous acid pH 1.5 and to heparinlyase I (data not shown). Importantly, none of these peaks wererecovered from control cultures of unstimulated BV2 cells (FIG. 4B).

Encephalotoxin as CSF Biomarker for Neurodegenerative Disease

Using ventricular CSF from rapid autopsy cases, encephalotoxin wasdetermined to be present in high concentrations in all CSFs from ADcases (confirmed by pathology), but not in cases from age-matchednormals or ALS (FIG. 5). Importantly, lumbar CSF taken from subjectswith a clinical diagnosis of probable AD also showed a striking pattern,with very high Encephalotoxin concentrations measured as ED₅₀s ofbetween 0.1 to 5 μl.

A research protocol was established to evaluate samples not only fromelderly subjects with cognitive impairment, but also from other groupsseen by our clinic neurologists. The latter populations consisted ofvarious diagnostic categories, with the largest groups suffering fromheadache variants, multiple sclerosis, or non-AD dementia (vascular,trauma). [Neurotoxin assays on these latter populations were performedwith subject consent on remnant aliquots of CSF acquired for otherclinical indications.] Data obtained thus far from subjects show thatall patients with probable AD have high levels of neurotoxin, with ED₅₀sfor equivalent CSF volumes ranging from 0.5 to 15 μl (note that lowerED₅₀ volumes indicate higher toxin concentrations); elderly subjectswith MCI had ED₅₀ of between 50 and 200 μl. The non-parametricKruskal-Wallis one-way ANOVA for ranks showed neurotoxin levelssignificantly differed (as measured by ED₅₀s) among tested diseasegroups (probable AD, MCI, non-AD dementia, headache, and MS;p=0.000001). The Kruskal-Wallis multiple-comparison test showed thatboth AD and MCI neurotoxin levels were significantly greater than theselevels found in MS, headache, or non-AD dementia (p<0.02 for allcomparisons).

Overall, these observations revealed several important trends. First,subjects with probable AD had the highest toxin concentrations, fallingwithin a narrow range, similar to that of ventricular CSF from ADautopsy cases. Second, severe cognitive impairment or dementia secondaryto non-inflammatory mechanisms (vascular, post-trauma) did not showdetectable amounts of encephalotoxin in the CSF. [While neurotoxin canbe found in tissues damaged acutely after stroke or trauma, theseneurotoxin levels dissipate as the acute inflammatory responsedissipates (about 3 to 7 days post injury; Giulian et al. (1990) Ann.Neurol., 27: 33-42; Giulian et al. (1993) Stroke, 24: 84-93; Giulian(1993) Glia, 7: 102-110)]. And third, there appeared to be a trend ofMCI subjects showing significant amounts of encephalotoxin, but only1/10 to 1/100 as much total toxic activity as found in AD CSFs (FIG. 5).

To determine whether oligosaccharides associated with encephalotoxinwere also found in human CSF, encephalotoxin was isolated from CSF byadsorption chromatography and treated with the same heparin lyases,nitrous acid treatments, and sulfatases as used for microglia culturemedia. Ventricular CSF from AD cases and lumbar CSF from probable ADsubjects demonstrated the same inactivation profiles (FIG. 1),indicating that encephalotoxin in human disease contained heparin-likeoligomers. Confirmation of the presence of such neurotoxicoligosaccharides came from anion-exchange HPLC, showing the presence ofa neurotoxic PEAK 38 recovered from microglial encephalotoxin fractions.There were similarities between the CSF samples from AD and MCI byanion-exchange profiles (PEAKS 38 and 48) with an additional PEAK 53 inthe MCI group (FIG. 6) as noted in microglial cultures (FIG. 4).

HPLC-profiles for ventricular cerebrospinal fluid of cases of definiteAD were nearly identical to lumbar fluid samples from volunteers withprobable AD (FIG. 7B) and from those with HAD (FIG. 7C). Enzymatictreatments by heparin lyase I and by N-sulfamindase eliminated all thesepeaks of neurotoxicity. Neuron-killing activity recovered byanion-exchange HPLC was insensitive to heparin lyase II (FIG. 7B),proteases, or heparin lyase III treatments (data not shown).

In order to survey the prevalence of neurotoxin production inneurological disorders, the cerebrospinal fluid of subjects from variousdisease populations was examined. Neurotoxin concentrations, expressedas CSF Neurotoxicity Index scores [expressed as equivalent volume of CSFwhich yields 50% of a total neuron killing effect in a standardized rathippocampal culture assay], show that only those subjects with definiteAD (postmortem diagnosis; n=7) or HIV-1 infection (n=52) had detectablelevels of CSF neurotoxin (FIG. 8A). Neurologic disorders that can elicitchronic reactive immune responses, such as multiple sclerosis (MS;n=20), amyotrophic lateral sclerosis (ALS; n=8), or neuropathies (n=14),had no CSF neurotoxin. Similarly, subjects with psychiatric illness(n=5), with headache (n=6), or a variety of other neurological diseases(n=21; including fungal meningitis and neurosyphilis) are free ofdetectable neurotoxin. And finally, CSF samples obtained from volunteersundergoing routine myelography (n=20) contained no neurotoxin activity.

Neurotoxicity Index values for CSF in cases of definite AD rangedbetween 1 and 10 whereas a broader distribution appeared for the HIV(+)population (0.1 to 1000). To investigate the wider distribution ofneurotoxin levels for the HIV-1(+) cohort, 7 coded lumbar CSF samplesfrom the HIV-1(+) volunteers who had undergone extensive medical,neurological, and neuropsychological evaluations were obtained throughthe Texas unit of the National Neuro-AIDS Tissue Consortium (Morello etal. (2001) Neuropath. Appl. Neurobiol., 27:326-335). Neurotoxins aredetected in those subjects with cognitive dysfunction (n=4) but not inthose found to have normal cognition (n=3; Fisher's Exact Test,p=0.028). Low CSF Neurotoxicity Index scores were detected in HIV(+)subjects with HAD (range from 0.1 to 4.0); high Index scores weredetected in HIV(+) subjects with little or no cognitive impairment(all >200), and intermediate Index scores (1.0 to 21.0) were associatedwith HIV(+) subjects identified with mild cognitive-motor disorder(MCMD;Working Group of American Academy of Neurobiology AIDS Task Force(1992) Neurology, 41:778; FIG. 8B). Significant differences between MCMD(median 7.3; mean+/−SE, 9.0+/−2.7; n=8) and HAD (0.1 median; 0.8+/−0.3;n=14) for Index values show a high confidence level (p=0.0001; KruskalWallis). The separation between HIV-1(+) subjects with MCMD group andthose without cognitive impairment (median 1000.0; mean900+/−99.7 μl;n=8) is also significant (p=0.001). The degree of HIV injury to the CNSreflects levels of CSF neurotoxin, implying causal relationships amongcognitive impairment, stage of brain pathology, and the production ofneuron poisons.

In order to determine whether neurotoxin levels also reflect cognitivedecline in the aged population, CSF was obtained from elderly volunteerswith Mild Cognitive Impairment of the amnestic type (MCI; objectivememory deficit, but without dementia; Bischkopf et al. (2002) ActaPsychiatr. Scand., 106:403-414; n=6), a condition of impaired memorythought to reflect an early stage of AD (DeKosky et al. (2003) Science,302:830). Comparison of subjects with MCI to elderly volunteers servingas controls (>70 years old and free of memory complaints; n=8) showedmarked differences between the groups (FIG. 8C). The Neurotoxicity Indexscores for MCI ranges from about 7 to 20 (median 10.0; mean 11.5+/−1.6;n=6) and are significantly lower than those measured for elderlycontrols (all >1000; n=8; Kruskal-Wallis; p=0.0005). Index scores forvolunteers with probable AD (defined by clinical criteria) show a rangeof values from 0.1 to 10 (median 1.7, mean 3.0+/−0.8; n=21). Probable ADand MCI values are also significantly different (p=0.0111;Kruskal-Wallis), further evidencing an association between levels of CSFencephalotoxin and stage of brain pathology underlying cognitivedysfunction. Importantly, other forms of dementia lacking chronic braininflammation, such as those secondary to trauma, alcoholism, or vascularinjury, produce little or no detectable CSF neurotoxin (median 1000;mean 933.0+/−66.6; n=12). These observations are in agreement with CSFencephalotoxin values found in autopsy-confirmed cases for definite AD(FIG. 8A) and for vascular dementia (FIG. 8A).

In order to classify groups according to CSF neurotoxin concentrations,discriminant analyses were applied to three diagnostic categories forHIV(+) subjects and three categories for the elderly. As shown in Table1, the CSF Neurotoxicity Index accurately predicts which HIV(+)volunteers will have little or no impairment in cognition (cut-off >100)from among those groups with MCMD (1-100) or HAD (<1). Similarly, theIndex correctly separates subjects with non-Alzheimer's dementia(cut-off >100) from the elderly with MCI or AD. A cut-off value of >100also predicts with 100% accuracy those elderly without memory complaints(see FIG. 8C).

TABLE 1 Cut-off Values for CSF Neurotoxicity Index According to DiseaseCategory A. Neurotoxicity Cut-off Values Diagnostic Group >100 1-100 <1HIV (+) unimpaired 100% 0% 0% (n = 11) Mild Cognitive 0% 100% 0% MotorDysfunction (MCMD) (n = 9) HAD (n = 14) 0% 21% 79% B. NeurotoxicityCut-off Values Diagnostic Group >100 >4-100 <4 Non-AD dementia 100% 0%0% (n = 20) MCI (n = 6) 0% 100% 0% AD (n = 20) 0% 33% 67%CSF Encephalotoxin as a Biomarker for Progression of Disease Pathology

Data from 164 subjects showed that all patients with AD have high levelsof neurotoxin in the CSF with ED₅₀s for equivalent CSF volumes rangingfrom 0.5 to 15 μl. Elderly subjects with mild cognitive impairment hadlevels between 50 and 200 μl. Subjects with various other neurologicaldisorders, including neurodegenerative diseases, had no detectabletoxicity (ED₅₀s>1000 μl); vascular and post-trauma non-AD dementia alsohad no toxic activity. HIV-1 (+) subjects demonstrated a wide range oftoxin concentrations (ED₅₀s ranging from 0.6 μl to >1000 μl).

CSF from 40 HIV-1 (+) individuals was examined. The level of toxicitywas associated with the degree of cognitive impairment. For example,HIV-1 (+) subjects with normal cognition showed ED₅₀s>1000 μl, whilethose with moderate to severe cognitive defects produced neurotoxinlevels of 0.6 to 5 μl, similar to the range found for AD subjects withestablished dementia. HIV-1 (+) subjects with mild to moderate cognitiveimpairments had intermediary levels of CSF neurotoxin with ED₅₀s rangingfrom 10 to 300 μl.

The Neurotoxicity Index in a variety of diagnostic groups was measuredand compared against definite AD (n=7; defined by neuropathologicdiagnosis using ventricular CSF obtained post mortem). As shown in FIG.8A, there is a striking difference between AD and other diagnosticcategories lacking measurable toxin (Index scores of 1000), thusevidencing the value of the Neurotoxicity Index across a broadpopulation. Furthermore, as shown in FIG. 9, CSF encephalotoxin levelsare clearly different among elderly without memory complaints or non-ADdementia (vascular, post traumatic, neurosyphillis) when compared to MCI(with amnestic features) or probable AD populations (using NINCDS-ADRDAdiagnostic criteria). Discriminant analyses (Table 1B) establishedcut-off Neurotoxicity Index values for AD at <4 and for MCI at 4 to 100,providing the ability to correctly assign diagnosis based upon toxinvalues for MCI or probable AD against other groups. The underlyingpathological process advances as a subject moves from a pre-symptomaticstate to mild impairment (MCI with a 1.5 SD drop below norms of astandardized memory test) and then to a more advanced stage withdementia (AD with a 2 SD drop below norms in memory and at least oneother domain). Earlier stages of disease prior to significant memoryloss (stages before diagnosis of MCI) involve the neuron-damaging immunecascade which is detectable by the presence of CSF encephalotoxin. Thissubclinical stage is the prodromic phase of AD pathology.

Correlation between toxin levels and clinical manifestations of diseaseprogression has been elucidated. MCI and mild AD subjects (MMSE>20;CDR<1) having CSF encephalotoxin were subjected to a detailedneuro-cognitive battery. Simple linear regression analyses were carriedout comparing Neurotoxicity Index values with T scores from sets ofstandardized tests representing major cognitive domains. (T scores arenormalized to 50 with 10 as SD; raw scores are adjusted for age, gender,ethnicity, and education level). As shown in Table 2, a highlysignificant correlation exists between Index scores and abnormal memory;that is, higher concentrations of toxin are found in those subjects withgreater memory deficits while other cognitive domains (abstraction,language, processing speed) are not.

TABLE 2 Cognitive Domain p = corr coef = Executive Function WisconsinCard Sort NS Trails B NS Memory/Learning Hopkins Verbal 0.007 0.784WMS-III 0.001 0.800 Information Processing digit symbol NS symbol searchNS Trails A NS Language FAS NS

Table 3 compares CSF Neurotoxin Index values and T scores for specificcognitive tests among HIV-1(+) volunteers (n=33). Confidence levels arebased upon linear regression analyses and show that cognitive defectswith domains of attention/information processing and learning/memory areclosely associated with the amount of CSF encephalotoxin, while languageand motor function are not. Prior to analysis, the Neurotoxicity Indexwas log transformed so that data would follow an approximate normaldistribution.

TABLE 3 Confidence Correlation Cognitive Domain Test Level (p=)Coefficient Abstraction/Problem Solving Visual Reasoning Wisconsin CardSort 0.010 0.462 Visual-Motor Trails Making B 0.003 0.514 SequencingLanguage Verbal Fluency FAS NS Learning and Memory Auditory Word ListHopkins Verbal 0.001 0.529 Learning Test Visual Simple Brief VisualMemory 0.001 0.531 Figures Test Word Recall Hopkins-Delayed 0.009 0.444Recall Figure Recall Brief Visual - 0.002 0.523 Delayed RecallAttention/Information Processing Auditory Series PASAT 0.000 0.735Number-Symbol WAIS III Digit 0.014 0.427 Translation Symbol VisualPatterns WAIS III Symbol 0.000 0.574 Search Visual-Motor Trails Making A0.011 0.442 Scanning Motor Abilities Psychomotor Grooved Pegboard NSSpeed/DexterityExamine Effects of Suppressive Agents for Microglia Upon CSFEncephalotoxin Levels

Use of encephalotoxin as a biomarker for monitoring drug treatment anddisease progression was examined in a 6-week double-blind randomizedstudy comparing several drugs against placebo with the primary endpointas change in encephalotoxin levels in the CSF. Despite the masking ofgroup assignments, a striking pattern was identified, as shown byrepresentative data in FIG. 10. Although some subjects receiving codeddrugs showed reduction in toxin levels by about 10-fold, such decreasesdid not shift subjects into the range of Index scores found among normalelderly (that is, Index scores remained below the MCI cut-off values of100). These data suggested that none of the active drugs used in thistrial were adequately dosed to provide complete neuroprotection. Thepersistence of significantly abnormal encephalotoxin concentrations madeit unlikely that a single drug trial would alter the clinical course ofAD.

A secondary endpoint was used to assess the ability of drug treatmentsto reduce Aβ-induced toxicity in cultured blood monocytes. It was foundin animal studies that blood mononuclear phagocytes reflect brainmicroglial responses to Aβ. Accordingly, drug responses in cultures ofblood monocytes having a baseline toxicity measure in enrolled subjectsprior to drug treatments were examined after entry into the maskedsingle drug trial. Study of 76 monocyte samples with measurement ofAβ-induced toxicity have shown the following:

-   -   1) in some cases a single drug (identity masked) completely        suppress Aβ-activation of blood monocytes;    -   2) single drugs that suppress blood monocytes offer only a        partial inhibition of CSF encephalotoxin levels;    -   3) ex vivo studies using blood monocytes from subjects without        evidence of drug suppression demonstrated exquisite sensitivity        to DAP/HCQ combinations at 1/10 doses.

The disclosure of each patent, patent application and publication citedor described in this document is hereby incorporated herein byreference, in its entirety.

Various modifications of the invention in addition to those shown anddescribed herein will be apparent to one skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

1. A method of detecting chronic neuroinflammation of the brain associated with cognitive impairment and neurodegeneration in a subject comprising detecting an encephalotoxin in a biological sample of said subject, wherein said step of detecting comprises comparing light absorbance of said biological sample in the presence of an encephalotoxin inactivator to light absorbance of said biological sample in the absence of said encephalotoxin inactivator, an increased absorbance in the absence of said encephalotoxin inactivator being indicative of said chronic neuroinflammation, and the encephalotoxin being an oligosaccharide comprising at least one glucosamine having N-sulfation and O6-sulfation and lacking peptide bonds.
 2. The method of claim 1 wherein said light absorbance is measured at a wavelength of 232 nanometers.
 3. The method of claim 1 wherein the encephalotoxin inactivator is heparin lyase I, N-sulfaminidase, glucosamine-6-sulfatase, or nitrous acid solution.
 4. The method of claim 1 wherein said chronic neuroinflammation is associated with HIV-1-associated dementia (HAD), neuro-AIDS, Creutzfeldt-Jakob Disease, Mild Cognitive Impairment, prion disease, minor cognitive/ motor dysfunction, or Alzheimer's disease (AD).
 5. A method of monitoring treatment of chronic neuroinflammation of the brain associated with cognitive impairment and neurodegeneration in a subject comprising comparing encephalotoxin level in a first and second biological sample of said subject, wherein said first biological sample is taken from said subject at an earlier timepoint than said second biological sample, wherein said second biological sample is taken from said subject following treatment, and wherein said encephalotoxin level is measured by light absorbance of said biological sample, an increased absorbance of said second biological sample being indicative of progression of said chronic neuroinflammation, the encephalotoxin being an oligosaccharide comprising at least one glucosamine having N-sulfation and O6-sulfation; and said encephalotoxin lacking peptide bonds.
 6. The method of claim 5 wherein said light absorbance is measured at a wavelength of 232 nanometers.
 7. The method of claim 5 wherein said first biological sample is taken from said subject following said treatment.
 8. The method of claim 5 wherein said chronic neuroinflammation is associated with HIV-1-associated dementia (HAD), neuro-AIDS, Creutzfeldt-Jakob Disease, Mild Cognitive Impairment, prion disease, minor cognitive/ motor dysfunction, acute stroke, acute trauma, or Alzheimer's disease (AD).
 9. The method of claim 5 wherein said subject is human, primate, bovine, equine, canine, feline, porcine, or rodent.
 10. The method of claim 5 wherein said subject is human.
 11. A method of monitoring progression of chronic neuroinflammation of the brain associated with cognitive impairment and neurodegeneration in a subject comprising detecting an increase in encephalotoxin level in said subject over time, wherein said step of detecting comprises measuring an increased light absorbance of an encephalotoxin in a first biological sample of said subject relative to light absorbance of an encephalotoxin of a second biological sample of said subject, wherein said second biological sample is taken from said subject before said first biological sample, and wherein said encephalotoxin comprises an oligosaccharide comprising at least one glucosamine having N-sulfation and O6-sulfation, and lacks peptide bonds, said increased light absorbance being indicative of progression of said chronic neuroinflammation.
 12. The method of claim 11 wherein said chronic neuroinflammation is associated with HIV-1-associated dementia (HAD), neuro-AIDS, Creutzfeldt-Jakob Disease, Mild Cognitive Impairment, prion disease, minor cognitive/ motor dysfunction, or Alzheimer's disease (AD).
 13. The method of claim 11 wherein said light absorbance is measured at a wavelength of 232 nanometers.
 14. A method of detecting an encephalotoxin in a biological sample of a subject, comprising comparing light absorbance of said biological sample in the presence of an encephalotoxin inactivator to light absorbance of said biological sample in the absence of said encephalotoxin inactivator, an increased absorbance in the absence of said encephalotoxin inactivator being indicative of said encephalotoxin, the encephalotoxin being an oligosaccharide comprising at least one glucosamine having N-sulfation and O6-sulfation, and lacking peptide bonds.
 15. The method of claim 14 wherein said light absorbance is measured at a wavelength of 232 nanometers.
 16. The method of claim 14 wherein the encephalotoxin inactivator is heparin lyase I, N-sulfaminidase, glucosamine-6-sulfatase, or nitrous acid solution.
 17. A method of detecting a neurological disease in a subject, said method comprising: determining a neurotoxicity index of cerebrospinal fluid of said subject, wherein the neurotoxicity index is equivalent to the volume in microliters of cerebrospinal fluid necessary to result in 50% killing of neurons in vitro relative to neuron survival in the presence of encephalotoxin inactivator-treated cerebrospinal fluid sample, wherein the encephalotoxin inactivator is heparin lyase I, N-sulfaminidase, glucosamine-6-sulfatase, or a nitrous acid solution, and wherein a neurotoxicity index of 100 or less is indicative of said neurological disease at a level associated with clinical diagnosis of disease.
 18. A method for monitoring change of neurological disease in a subject, said method comprising: determining a neurotoxicity index of a first cerebrospinal fluid sample of said subject and a neurotoxicity index of a second cerebrospinal fluid sample of said subject, wherein the neurotoxicity index is a calculated value determined by a bioassay comprising 1) contacting a cerebrospinal fluid sample of said subject with neurons in vitro, and 2) comparing neuron survival in the presence of said cerebrospinal fluid sample to neuron survival in the presence of encephalotoxin inactivator-treated cerebrospinal fluid sample, wherein the encephalotoxin inactivator is heparin lyase I, N-sulfaminidase, glucosamine-6- sulfatase, or a nitrous acid solution and wherein the neurotoxicity index is equivalent to the volume of cerebrospinal fluid sample necessary to result in 50% killing of neurons relative to neuron survival in the presence of encephalotoxin inactivator-treated cerebrospinal fluid sample, wherein said second cerebrospinal fluid sample is taken at a later time point than said first cerebrospinal fluid sample, wherein a decrease in said neurotoxicity index of said second cerebrospinal fluid sample relative to said neurotoxicity index of said first cerebrospinal fluid sample is indicative of progression of said neurological disease, and wherein an increase in said neurotoxicity index of said second cerebrospinal fluid sample relative to said neurotoxicity index of said first cerebrospinal fluid sample is indicative of regression of said neuroinflammation.
 19. The method of claim 18 wherein one of said cerebrospinal fluid samples is taken during the prodromic phase of said neurological disease.
 20. A method of monitoring treatment of neurological disease in a subject, said method comprising: determining a neurotoxicity index of a first cerebrospinal fluid sample of said subject and a neurotoxicity index of a second cerebrospinal fluid sample of said subject, wherein the neurotoxicity index is a calculated value determined by a bioassay comprising 1) contacting a cerebrospinal fluid sample of said subject with neurons in vitro, and 2) comparing neuron survival in the presence of said cerebrospinal fluid sample to neuron survival in the presence of encephalotoxin inactivator-treated cerebrospinal fluid sample, wherein the encephalotoxin inactivator is heparin lyase I, N-sulfaminidase, glucosamine-6-sulfatase, or a nitrous acid solution, and wherein the neurotoxicity index is equivalent to the volume of cerebrospinal fluid sample necessary to result in 50% killing of neurons relative to neuron survival in the presence of encephalotoxin inactivator-treated cerebrospinal fluid sample, wherein said second cerebrospinal fluid sample is taken following said treatment and at a later time point than said first cerebrospinal fluid sample, wherein a decrease in said neurotoxicity index of said second cerebrospinal fluid sample relative to said neurotoxicity index of said first cerebrospinal fluid sample is indicative of progression of said neurological disease, and wherein an increase in said neurotoxicity index of said second cerebrospinal fluid sample relative to said neurotoxicity index of said first cerebrospinal fluid sample is indicative of successful treatment.
 21. The method of claim 17, claim 18, or claim 20 wherein said neurological disease is HIV-I-associated dementia (HAD), neuro-AIDS, Creutzfeldt-Jakob Disease, Mild Cognitive Impairment, prion disease, minor cognitive/motor dysfunction, or Alzheimer's disease (AD) or the prodromic phase of HIV-I-associated dementia (HAD), neuro-AIDS, Creutzfeldt-Jakob Disease, Mild Cognitive Impairment, prion disease, minor cognitive/motor dysfunction, or Alzheimer's disease (AD).
 22. A method of detecting an encephalotoxin in a biological sample of a subject comprising: A) contacting a biological sample of said subject with neurons, wherein said biological sample is cerebrospinal fluid, spinal cord tissue, or brain tissue, and B) comparing neuron survival in the presence of said biological sample treated with encephalotoxin inactivator relative to neuron survival in the presence of said biological sample not treated with said encephalotoxin inactivator, wherein a decrease in neuron survival in the absence of said encephalotoxin inactivator treatment relative to neuron survival in the presence of said encephalotoxin inactivator treatment is indicative of said encephalotoxin, and wherein the encephalotaxin inactivator is heparin lyase I, N-sulfaminidase, glucosamine-6-sulfatase, or a nitrous acid solution.
 23. The method of claim 17, 18, 20, or 22 wherein said subject is human, primate, bovine, equine, canine, feline, porcine, or rodent. 